Biological membranes regulate fluxes of water and solutes over a 10,000-fold permeability (P) range, from apical membranes of low P barrier epithelia to high P membranes which contain aquaporins (AQPs) or urea transporters UT's). This grant addresses two fundamental physiological questions: Aim #1. What are the lipid and protein structural features of barrier epithelial apical membranes that reduce their P's to water and to other small solutes such as urea, H+, and NH3? Aim #2. How do the structures of AQP's and UT's determine their ability to mediate the fluxes of water and small solutes across membranes? Question #1 of Aim #1 will focus on the lipid determinants of apical membrane barrier function. We will obtain the best structural information (volume and thickness of the hydrocarbon ore, hydration radius of head groups, elastic and compression moduli) available and we will measure the water content as a function of depth within the bilayer for 6 phospholipids with and without 50% cholesterol. We will then measure P's to water, urea, glycerol, NH3 and H+. Comparing structural parameters and water content with P's will define the mechanisms by which lipids can limit permeation. Question #2 of Aim #1 will examine the role of uroplakins, which are integral membrane proteins, in the barrier function of urothelia. We will examine P's, ultrastructure, membrane trafficking in response to stretch and emptying, and the ability of these proteins to enhance the unstirred layer of the epithelium using background strain mice and mice in which uroplakins have been ablated. Question #1 of Aim #2 will compare molecular dynamics (MD) simulations of H2O, D2O, CO2 and H2S conductance across two AQP's for which the structure of the pore has been defined (AQP1 and the bacterial AQP, AQPZ) to actual measurements of these conductances, permitting us to enhance the true predictive ability of these simulations. Question #2 of Aim #2 will exploit a newly developed expression system, Xenopus oocyte plasma membrane vesicles, to examine in detail the function of renal UT's (UT-A's) and the red cell UT (UT-B). Because these UT's share a great deal of sequence homology, detailed functional studies will relate amino acid sequence to transport function. We will define the mechanism of cAMP regulation of UT-A function. We have also obtained two bacterial UT's, and will use these for functional studies and efforts at reconstitution and structural studies. Building on substantial prior progress, this competing renewal seeks to define at a molecular level the mechanisms by which water and other small molecules cross barrier membranes, AQP's, and UT's. [unreadable] [unreadable] [unreadable]